We have developed yeast vacuoles as a paradigm of membrane fusion in endocytic and exocytic traffic. Each stage of this traffic, from yeast to humans, uses similar Rab, effector complex, SNAREs, and SNARE chaperones, but studies of yeast vacuole fusion have unique technical advantages: superb genetics, genomics, and biochemistry of baker's yeast, ease of visualizing the vacuole and purifying it, and the rapid and quantitative assays which we've developed for fusion and its subreactions. In addition to studying vacuole fusion in vivo and with the purified organelle in vitro, we have developed in this funding period a reconstitution of proteoliposome (RPL) fusion with all-purified components: the 4 vacuolar SNAREs, the Ypt7p Rab GTPase, its effector complex HOPS (in which the vacuolar SM protein Vps33p is one of 6 subunits), the SNARE disassembly chaperones Sec17p/Sec18p, 9 physiological vacuolar lipids, and ATP. This system has revealed basic mechanistic features of fusion which underlie each aim of our proposal: (i) The fusion pathway proceeds through defined stages;we will use our published assays of tethering and trans-SNARE complex formation, and will exploit our fluorimetric assays of content mixing, lipid mixing, permeability, and lysis, using agents that interrupt fusion to seek hemifusion (partial lipid mixing without content mixing). (ii) Diverse vacuolar lipids are needed for rapid fusion, beyond simply forming a bilayer;we will explore which are needed for peripheral membrane protein binding, tethering, trans-SNARE complex assembly, and the lipid rearrangements of hemifusion and fusion. (iii) Fusion catalysts undergo regulated cycles: 1. HOPS and Sec17/18p synergistically support fusion as SNAREs cycle between cis-complexes, the uncomplexed state, and trans-complexes, 2. Gyp7p and Ccz1/Mon1 catalyze the Rab Ypt7p cycling between GTP- and GDP-bound states, and 3. the vacuolar kinase Yck3p and unknown phosphatase(s) regulate HOPS phosphorylation. These 3 cycles are likely related, and our unique tools will allow exploration of these relationships. Understanding the vacuolar HOPS complex, discovered and studied in this work, underlies studies of human arthrogryposis, renal dysfunction, and cholestasis syndromes, caused by mutations in the human VPS33 gene. Human HOPS has recently been shown to have a unique and central role in Marburg and Ebola virus invasion of human cells. The proposed studies are thus important for both basic and clinical science.

Public Health Relevance

Membrane fusion is essential for processes as diverse as cell growth, hormone secretion, and neurotransmission, but its mechanism is highly conserved from yeast to humans. By studying the fusion of yeast vacuoles (lysosomes) at each level, defining the needed proteins and lipids, purifying them, and reassembling them into defined reactions which bear on all facets of the mechanism, we have discovered fundamental components whose human homologs are required for viral infection (Ebola and Marburg virus) and underlie certain inherited human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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Dartmouth College
Schools of Medicine
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Zick, Michael; Stroupe, Christopher; Orr, Amy et al. (2014) Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. Elife 3:e01879
Zick, Michael; Wickner, William T (2014) A distinct tethering step is vital for vacuole membrane fusion. Elife 3:e03251
Karunakaran, Vidya; Wickner, William (2013) Fusion proteins and select lipids cooperate as membrane receptors for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vam7p. J Biol Chem 288:28557-66
Stroupe, Christopher (2012) The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex. Biochem J 443:205-11
Xu, Hao; Zick, Michael; Wickner, William T et al. (2011) A lipid-anchored SNARE supports membrane fusion. Proc Natl Acad Sci U S A 108:17325-30
Zucchi, Paola C; Zick, Michael (2011) Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol Biol Cell 22:4635-46
Xu, Hao; Wickner, William (2010) Phosphoinositides function asymmetrically for membrane fusion, promoting tethering and 3Q-SNARE subcomplex assembly. J Biol Chem 285:39359-65
Xu, Hao; Jun, Youngsoo; Thompson, James et al. (2010) HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion. EMBO J 29:1948-60
Hickey, Christopher M; Wickner, William (2010) HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly. Mol Biol Cell 21:2297-305
Stroupe, Christopher; Hickey, Christopher M; Mima, Joji et al. (2009) Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components. Proc Natl Acad Sci U S A 106:17626-33

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